There is without doubt considerable interest in simple apparatus for the reliable, selective detection of gases. Such gases can either be present directly as components of a gaseous mixture, such as air, or from a biological tissue, such as from a photosynthesizing plant leaf, or e.g. from a biological reaction. The biologically active systems can be living organisms, such as bacteria or biologically active extracts from biological samples, e.g. enzymes. The biologically active system is generally suspended in the liquid, namely water, or is immobilized on a carrier, e.g. a plastic, metal or glass surface.
An apparatus for the detection of gases dissolved in a liquid and formed e.g. by a biological reaction are described in Swiss Patent Application 572/84-1 of Feb. 7th 1984.
However, there are also microbiological systems such as enzymes and bacteria, which are reactive with the air. They either can be present in aerosol form, or, as stated, can be immobilized on a carrier.
If a substrate is added, which substrate can be decomposed by the biologically active system, then gaseous products can form. The gas produced is then a measure of the activity of the biological organism or the bioactive extract.
On the basis of the gas production, information can also be obtained on the concentration of the substrate. As in general biological reactions take place in a highly specific, selective manner, the possibility arises of obtaining, on the basis of the determination of the gases produced, clear and qualitative information on the substrates. Equipment operating according to this principle are covered by the term biosensors. The problem arises of detecting the reaction product, i.e. to produce an electric signal, whose quantity is related to the concentration of the dissolved gases.
For the detection of gases, it is possible on the one hand to use chemical methods, such as titration, calorimetry, gravimetric determination of precipitates, etc. and on the other handpurely physical methods, such as optical spectroscopy, mass spectrometry, etc.
Admittedly chemical methods are generally simple with regards to the equipment, but as a result of the complexity of the reaction sequence, e.g. the reagent supply, separation by filtration, etc., they are less suitable for automatically controlled measurements.
It may also be necessary to carefully separate the apparatus where the biological decomposition takes place, the so-called bioreaction from the chemical reactor, in order to prevent poisoning by chemicals of the bioactive, systems.
Another unfavourable point in chemical determinations is the time taken for the chemical reaction and for the test specimen or sample preparation.
Therefore preference is given to physical determination of gaseous biological reaction products. Unfortunately corresponding methods are usually linked with high equipment expenditure, particularly in the case of spectroscopic methods. However, it must be stressed that optical-spectroscopy methods are characterized by high reliability and selectivity, so that further comment will be made thereon.
The following spectral ranges are worthy of consideration for the optical-spectroscopic detection of biological products: ultraviolet and visible range (180-800 nm), and infrared range (0.8 to 20 .mu.m).
In the ultraviolet and visible spectral ranges, the molecules in question generally have broad-band and less specific absorption structures. Thus, apart from exceptions, this spectral range is generally not very important. However, with regards to selective detection, considerable importance is attached to the infrared spectral range. Particularly in the range of 10 .mu.m, many organic molecules have very specific absorption lines (fingerprint range).
An important disadvantage of infrared spectroscopic analytical method is that it is scarcely possible to perform tests in aqueous solutions, because the water has very strong, all-surpassing absorption lines distributed over the entire important infrared spectral range. Thus, for infrared tests, organic substances are usually dissolved in organic solvents or processed to suspensions. This possibility is generally unsuitable for investigating substances obtained from biological reactions, because, apart from a few exceptions, biological reactions take place in water and consequently the reaction products also exist in the form of an aqueous solution or suspension.
Thus, infrared spectroscopic detection methods almost unavoidably require separation of water from the reaction product. The latter can be dried and either, as stated, mixed with an organic solvent or can be mixed with an inorganic salt, such as KBr, and pressed into a pill.
The separation of the reaction product from water is time-consuming and complicated. This method would certainly be unsuitable for automatic detectors. As a result of the gas/liquid equilibrium, it would be relatively easy to separate a reaction product with a high vapour pressure, at least partly from the water. It is merely necessary to prevent the problems caused by water vapour.
There are two different methods available for optical spectroscopic detection of gases and vapours, on the one hand the direct extinction method and on the other the photoacoustic method.
The extinction method comprises measuring the light intensity before (Io) and after (I) the gas absorption cell. Conclusions can be drawn regarding the concentration of the light-absorbing gas from the difference (Io-I) of the two measurements. This method permits analysis in flowing gas. However, it is only suitable for determination of relatively high gas concentrations, unless very considerable cell lengths or multiple reflection cells can be accepted. However, in the latter case it is necessary for the incident light beam to be narrowly focused (although if necessary the divergence of the beam can be counteracted by a special mirror shape), and on the other hand the mirror system must be very accurately adjusted. Thus, multiple reflection cells are generally very complicated and costly.
If a reliable measurement is to be performed, then the light attenuation (Io-I)/Io must move within certain limits. A lower limit of 1% and an upper limit of 99% means that no great demands must be made on the stability of the light source and light detectors. The optical path length of the gas cell must be adapted to these limits.
For the detection of low concentration, atmospheric gas contaminants, such as CO or CH.sub.4, the photoacoustic gas detection method has proved very satisfactory. This method consists of the detection by a microphone of pressure changes, which occur in a gaseous mixture on absorbing monochromatic, intensity-modulated light (mainly infrared radiation) due to a gas component, such as is described in the article by L. G. Rosengren (Appl. Optics, 14, 1968, 1975. For this purpose, generally intense, matchable infrared lasers, together with highly sensitive capacitor microphones are used. The method is very sensitive, e.g. L. B. Kreuzer, J. App. Phys, 42, 1934, 1974 was able to detect methane in nitrogen in a concentration of 10 ppb (10E-8) with the aid of a 16 mW laser.
On accepting much lower requirements regarding the sensitivity of gas detection, considerable simplification of the detection system is possible. In particular the costly, matchable infrared laser can be replaced by a simple system having an incandescent body and a narrow-band interference filter. Admittedly of late M. J. D. Low and G. A. Parodi, Infrared Phys., 20, 333, 1980 have described an infrared spectrometer based on the photoacoustic effect, in which an incandescent pin is used in place of the laser. However, as a result of its weak intensity, this source has not proved very satisfactory when combined with a grating monochromator for a photoacoustic infrared spectrometer. The replacement of the grating monochromator by an interference filter leads to advantages regarding the intensity measurement, but the flexibility and accuracy suffer.
Our own measurements have shown that very good results can be obtained in connection with the sensitivity thus, when using a thermal radiation source mechanically modulated at 5.6 W electric power and an interference filter as the monochromator (light power 2.6 E-5 AVW cm) CO.sub.2 could be reliably detected in N.sub.2 with a concentration of 1.5 ppm, as described in the article by O. Oehler, D. Marek and A. Fries, (Helv. Phys. Acta, 54, 631, 1981).
In a certain sense it is surprising that this method can operate without lasers. However, it is pointed out in this connection that in general laser radiation can only excite a single sharp rotational line of the complex vibration-rotation spectrum of a low molecular gas. The combination of a thermal radiation source and interference filter makes it possible to excite the complete absorption band system. As the photoacoustic effect is proportional to the total light power absorbed, it is interesting that a good result can still be obtained when using a weak intensity broad-band light source.
However, it must be borne in mind that the light can be concentrated very efficiently in a detector gas cell of small volume V, because one third L/V of the light power L absorbed in the cell is decisive for the size of the photoacoustic signal. The proportionality between the photoacoustic cell and the light intensity mesns that high demands cannot be made on the intensity stability of the light source, contrary to the situation in the extinction method.
In one category of gas analyzers based on the photoacoustic effect, the infrared laser has been successfully replaced by an incandescent body. These consist of the so-called, non-dispersive photoacoustic gas analyzer systems, of which numerous different constructions exist. Reference is made in this connection to German Pat. No. 2751047 of O. H. Blunck and No. 2748089 of U. Deptolla and F. Fabinski.
In the case of these non-dispersive systems, it is not the absorptive light quantity absorbed by the gas component adapted to the filter which directly gives rise to the microphone signal, but instead the difference of the light attenuation between the sample gas and a reference gas is selectively measured by means of a split up light beam. The gas-selective light intensity difference measurement is determined with a photoacoustic difference measuring cell, which is filled with the gas of the component being tested. This construction has the advantage that no monochromatic radiation is required. However, a corresponding apparatus only makes it possible to determine a single gas type. Moreover, such systems have a complicated construction, because in conjunction with the difference signal measurement, the two light beam fractions have to be very accurately compensated.
In summary it can be stated that optical-spectroscopic gas detection methods almost always require considerable effort and expenditure. However, the intention is to provide a simple detection apparatus for gases dissolved in liquid and this requirement is met by the photoacoustic measuring principle using a thermal source, as described in the article by O. Oehler and D. Sourlier (Helv. Phys. Acta, 55, pp. 594-597, 1982).
The photoacoustic effect is based on the fact that measurement takes place through the sound field formed in the gas cell through the absorption of intensity-modulated light radiation. Thus, a photoacoustic measuring apparatus is sensitive from the outset to acoustic disturbances. It is therefore unavoidable that corresponding sound-attenuating measures are taken.
During the measurement it must on the one hand be ensured that the light absorption-resulting sound signal is not weakened by the escape of gas from the photoacoustic cell and on the other hand the penetration of external space sound, which could increase the noise level of the measurement, is prevented.
If operation takes place with a high modulating frequency, it is possible to make do with simple flow-resisting elements, such as diaphragms and the like. However, it is unavoidable in the case of low modulating frequencies to use efficient sound attenuating means. However, great importance is attached to the operation of a photoacoustic gas detector at low modulating frequencies, because corresponding equipment involves little expenditure. If necessary, the light source can even be thermally modulated by switching the light source current on and off, which certainly represents a simple and reliable solution, compared with light interruption, with a mechanical aid, e.g. a rotating diaphragm. Operation normally takes place with completely closed photoacoustic gas detector cells.
Very simple solutions for the acoustic separation of the leads are described in the patent applications of Oehler et al., PCT/CH82/00026 and PCT/CH83/00080. They are based on a hydrodynamic acoustic decoupling. They are based on the fact that air and liquid media have very different acoustic stiffnesses (h=E, E=modulus of elasticity and h=density) and consequently the acoustic power matching at the gas--liquid and liquid--gas interfaces is very poor. In the human ear, e.g. where the problem in question arises with sound transmission from the outer ear to the perilymph of the inner ear, the necessary good power matching is ensured by the auditory ossicle within the middle ear. The different acoustic stiffnesses of air and perilymph is taken into account by a mechanical transmission (transmission ratio 60:1).
One acoustic separating apparatus comprises a liquid-filled, gas washing bottle-like dipping tank being fitted on either side of the photoacoustic gas detector. By means of a limited overpressure, which can e.g. be produced by means of a diaphragm pump, gas is blown through the liquid and consequently the exchange of the gas in the cell is accomplished. Admittedly no photoacoustic measurement can be performed during this process. The typical noise occurring on blowing the gas through the liquid would disturb the measurement too much.
On switching off the pump, the liquid level stabilizes, so that acoustic decoupling is ensured. There is an acoustic attenuation of 40 to 55 db in the case of measurements in the 10 to 100 Hz frequency range.
A second construction of the acoustic separating apparatus comprises the separating liquid being held in narrow tubes by capillary forces. Under a limited overpressure, the liquid is expelled from the capillary tubes which ensures the gas exchange. On switching off the pump, the liquid flows back into the capillary tubes the valve being acoustically closed. The acoustic attenuation of this apparatus is high and values of 60 to 70 dB have been obtained in the frequency range of 10 to 100 Hz.
It is conceivable to suck off the medium to be detected, i.e. gas or vapour, and to supply it to the photoacoustic cell for optical-spectroscopic testing. Consideration can indeed be given to this method if the connecting means between the gas removal or gas collecting point and the detection cells can be made geometrically small, so that a rapid gas transfer is possible. However, a very close contact is sought, between the removal/collecting point and the gas detector.
Account has been taken of this in a further construction relating to acoustic separation in the photoacoustic cell. This third construction is described in Swiss Patent Application 572/84-1 of Feb. 7th 1984 and 2594/84 of May 28th 1984. The inner area of the photoacoustic gas detector is separated from the outside, constituting the gas collecting point, by a gas-permeable diaphragm. Despite its gas permeability, such a diaphragm provides a sufficiently large acoustic attenuation to ensure a photoacoustic measurement.
This arrangement leads to a particularly simple gas detector, in that the gas exchange between the gas collecting point and the photoacoustic gas detector takes place purely passively by gas diffusion through the diaphragm, instead of requiring a pump.
It has proved advantageous if no light from the test beam strikes the gas-permeable diaphragm. In the case of diaphragm illumination, even when an absorbing gas is absent in the detector room there is a large photoacoustic signal, which can be attributed to the light absorption in the diaphragm.
It is also pointed out that a gas-permeable diaphragm is not an ideal acoustical resistance, particularly if operation is to take place at low light modulating frequencies. In accordance with the calculations, with a 0.9 cm.sup.3 capacity photoacoustic cell separated by a gas-permeable diaphragm with a diameter of 0.5 cm, acoustic attenuation factors of 5 to 30 dB were obtained at 5 Hz, as a function of the diaphragm type.
Significantly better results can be expected on replacing the thin, flexible, gas-permeable diaphragm by a rigid, porous material such as e.g. a sintered product.
Thus, the photoacoustic signal is generally small and very noisy. These disadvantages can be counteracted by using an intense, intensity-modulated light source, as well as a very low-band electronic filter.
As the diaphragm-decoupled, photoacoustic gas detector is an extremely simple and inexpensive gas detection means, the light source and the control thereof, as well as the signal detection electronics must be made correspondingly simple. Such means are described in Swiss Patent Application 4249184-3 of September 6th 1984.
Patent application PCT/CH 83/00080 already describes a very efficient thermal light source. The hereinafter described invention is based on a photoacoustic gas detector, which is acoustically separated from the gas collecting point by a gas-permeable diaphragm or by a rigid porous material, such as e.g. a sintered product, as well as electronic means for the intensity modulation of the light source and for detecting the microphone signal.